Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport

Abstract

Background: Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT.

Results: fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33 degrees C), as previously reported. We find that in standard media containing approximately 300 microM calcium, fla10 cells lose flagella by deflagellation at 33 degrees C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants.

Conclusions: Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active disassembly is distinct from the constitutive disassembly which plays a role in flagellar length control.

Figure 1

Free flagella in Chlamydomonas media are indicative of deflagellation. Determination of free flagella under various conditions. Free flagella observed per 100 cells were counted after 0 and 6 hours at 20°C or 33°C, in either the presence (TAP medium or HEPES+Ca) or absence (HEPES buffer) of calcium, and the difference 6 h – 0 h was calculated. Each data point is the average of at least three experiments on independent cultures.

Figure 2

fla10 and fla2 average flagellar lengths decrease during incubation at 33°C. Cells were resuspended in pre-warmed buffer at t = 0 h. At least 70 cells were examined per timepoint. wild-type, fla2, fla10. (A) Incubation in HEPES, including contribution of bald and uniflagellate cells.(B) Incubation in HEPES+Ca, including contribution of bald and uniflagellate cells.(C) The same samples as (A), but averages exclude contributions of zero-length flagella.(D) The same samples as (B), but averages exclude contribution of zero-length flagella.

Figure 3

Scatter plots of fla10 timecourses in HEPES and HEPES+Ca. Data shown is the same as fla10 data in Figure 2. Each flagellum from at least 70 cells was measured, and the lengths plotted as the length of the longer flagellum (in μm) on the X-axis and the length of the shorter flagellum (in μm) on the Y-axis, such that each point in a graph corresponds to a single cell (except where two points overlap, such as at the origin). Insets show the percent of biflagellate (white), uniflagellate (grey), and bald (black) cells in each sample.

Figure 4

Scatter plots of fla2 timecourses in HEPES and HEPES+Ca. See Figure 3 legend for further details.

Figure 5

Scatter plots of wild-type timecourses in HEPES and HEPES+Ca. See Figure 3 legend for further details.

Figure 6

Double mutants of fla10 with fa1 or fa2 are slower to disassemble flagella at the restrictive temperature than fla10 single mutants. Scatter plots of 33°C timecourses for fla10, fla10-fa1 and fla10-fa2; plots prepared as described in the legend to Figure 3. TAP cultures were resuspended in pre-warmed TAP media at t = 0 h. No free flagella were seen in fla10-fa1 or fla10-fa2 cultures at any timepoint.

Figure 7

Acid shock causes resorption of mutants unable to deflagellate. At the indicated times post-acid shock, flagellar length distributions were determined and percents of cells with long flagella (flagella longer than 7 μm) were plotted.

Figure 8

Flagella: to have or to have not. Model for the co-regulation of flagellar assembly (mediated by IFT) and active flagellar disassembly by a common signal, X. Under conditions either permissive or nonpermissive for flagellar assembly, IFT and disassembly are proposed to be mutually antagonistic. Under conditions appropriate for flagellar assembly (Yes), the decision is made to activate IFT and block disassembly at the transition zone. Active IFT could provide a signal to inhibit active disassembly. When flagella are to be disassembled (No), IFT is inactivated. In the absence of IFT, an inhibitory signal would be removed, allowing disassembly to proceed. There may also be direct activation of disassembly.